The ISME Journal (2016) 10, 2048–2059
© 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16
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www.nature.com/ismej
ORIGINAL ARTICLE
Comparative genomics reveals new evolutionary and
ecological patterns of selenium utilization in bacteria
Ting Peng, Jie Lin, Yin-Zhen Xu and Yan Zhang
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences,
Shanghai, PR China
Selenium (Se) is an important micronutrient for many organisms, which is required for the
biosynthesis of selenocysteine, selenouridine and Se-containing cofactor. Several key genes
involved in different Se utilization traits have been characterized; however, systematic studies on
the evolution and ecological niches of Se utilization are very limited. Here, we analyzed more than
5200 sequenced organisms to examine the occurrence patterns of all Se traits in bacteria. A global
species map of all Se utilization pathways has been generated, which demonstrates the most detailed
understanding of Se utilization in bacteria so far. In addition, the selenophosphate synthetase gene,
which is used to define the overall Se utilization, was also detected in some organisms that do not
have any of the known Se traits, implying the presence of a novel Se form in this domain.
Phylogenetic analyses of components of different Se utilization traits revealed new horizontal gene
transfer events for each of them. Moreover, by characterizing the selenoproteomes of all organisms,
we found a new selenoprotein-rich phylum and additional selenoprotein-rich species. Finally, the
relationship between ecological environments and Se utilization was investigated and further verified
by metagenomic analysis of environmental samples, which indicates new macroevolutionary trends
of each Se utilization trait in bacteria. Our data provide insights into the general features of Se
utilization in bacteria and should be useful for a further understanding of the evolutionary dynamics
of Se utilization in nature.
The ISME Journal (2016) 10, 2048–2059; doi:10.1038/ismej.2015.246; published online 22 January 2016
Introduction
Selenium (Se) is an important trace element for
many organisms in the three domains of life; yet, it is
required only in small amounts. This element is
known primarily for its functions in redox homeostasis, and is recognized as a promising cancer
chemo-preventive agent. In addition, Se is required
for mammalian development, male reproduction and
immune function (Rayman, 2000; Hatfield et al.,
2006). To date, three biological forms of Se have
been identified: (i) selenocysteine (Sec, the 21st
amino acid), the major biological form of Se, which
is co-translationally inserted into selenoproteins
by recoding the UGA codon (Böck et al.,
1991; Stadtman, 1996; Hatfield and Gladyshev,
2002); (ii) 5-methylaminomethyl-2-selenouridine
(mnm5Se2U or SeU), which is located at the wobble
Correspondence: Y Zhang, Key Laboratory of Nutrition and
Metabolism, Institute for Nutritional Sciences, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, University
of Chinese Academy of Sciences, 294 Tai Yuan Road, Shanghai
200031, PR China.
E-mail: [email protected]
Received 2 June 2015; revised 28 October 2015; accepted
27 November 2015; published online 22 January 2016
position of the anticodons of some prokaryotic
tRNAs and may help base pair discrimination and/
or improve translation efficiency (Ching et al., 1985;
Wittwer and Ching, 1989); (iii) a Se-containing
cofactor (Se-cofactor) used by certain molybdenumcontaining enzymes in both bacteria and archaea
(Haft and Self, 2008; Zhang et al., 2008).
The decoding of UGA as Sec is an intriguing
example of alternative genetic decoding phenomenon. Alterations in genetic decoding are expected to
exist in almost all organisms (Baranov et al., 2015).
Many mRNAs have evolved special elements to alter
the meaning of specific codons or to result in
ribosomal frameshifting or even translational bypassing (Atkins and Baranov, 2010; Chin, 2014). The
mechanism of Sec biosynthesis and its insertion into
proteins has generally been elucidated in all three
domains of life (Low and Berry, 1996; Böck, 2000;
Thanbichler and Böck, 2002; Allmang et al., 2009).
In bacteria, this process requires an in-frame UGA
codon, a Sec insertion sequence (SECIS) element that
is a hairpin structure within the selenoprotein
mRNA immediately downstream of the Secencoding UGA codon, and several trans-acting
factors including Sec synthase (SelA), Sec-specific
elongation factor (SelB), Sec-specific tRNA[Ser]Sec
Comparative genomics of selenium utilization
T Peng et al
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(a unique tRNA whose anticodon matches the UGA
codon) and selenophosphate synthetase (SelD).
In eukaryotes and archaea, the biosynthesis of
Sec uses a similar mechanism as in bacteria.
However, additional genes such as genes encoding
the eukaryotic/archaeal Sec synthase, O-phosphoseryl-tRNA[Ser]Sec kinase and SECIS binding protein
SBP2 are needed for incorporation of Sec into
protein (Copeland and Driscoll, 2001; Xu et al.,
2007; Squires and Berry, 2008). In bacteria, the entire
Sec-decoding trait and selenoprotein genes could be
laterally transferred between evolutionarily distant
organisms to extend Se utilization (Romero et al.,
2005; Zhang et al., 2006); however, such event could
not be detected in multicellular eukaryotes such as
animals.
In many prokaryotes, the product of SelD protein,
selenophosphate, is also used for the biosynthesis of
SeU. The 2-selenouridine synthase (YbbB) is needed
during SeU utilization (Wolfe et al., 2004). In
addition, a new SelD-based Se utilization form was
reported in some bacteria, in which Se might be used
as a cofactor by molybdenum-containing hydroxylases via two gene products whose function is
unclear as yet: YqeB and YqeC (Haft and Self,
2008; Zhang et al., 2008). Thus, it appears that each
known Se utilization trait has unique genes, whereas
SelD is considered as a common signature for Se
utilization (Romero et al., 2005; Zhang et al., 2008).
In the recent decade, the majority of studies on Se
focused on investigation of new genes and pathways
involved in Se metabolism and the function of
selenoproteins. In spite that several comparative
genomics analyses of components of Se utilization
traits in completely sequenced bacterial genomes
have provided evidence for a highly diverse pattern
of species that use Se (Romero et al., 2005; Zhang
et al., 2006, 2008; Zhang and Gladyshev, 2010b; Lin
et al., 2015), it is still unclear how this trace element
is used by a subset of organisms. A more puzzling
question is whether the evolution of Se utilization
could be influenced by various ecological conditions. With the rapid increase in the number of
sequenced genomes in recent years, it would be
interesting to determine the general utilization trend
of different Se forms and the relationship between
environmental conditions and Se utilization.
In this study, we applied comparative genomics
approaches to more than 5200 sequenced bacterial
genomes to investigate Se utilization in this domain.
First, we analyzed the distribution of key components involved in different Se utilization traits and
generated a large phylogeny map of Se utilization in
bacteria. We also identified a subset of organisms
that had selD gene but lacked any of the known Se
traits, implying that a new SelD-based Se utilization
pathway is present in these organisms. Further
analysis of the whole set of selenoproteins (selenoproteomes) of all Sec-utilizing organisms showed the
presence of a new selenoprotein-rich phylum and
more selenoprotein-rich organisms in bacteria.
Finally, analysis of environmental factors of all
organisms revealed that different Se traits may favor
specific ecological conditions such as habitat, oxygen concentration and temperature. As a whole,
these data provide a better understanding of the
general trends of Se utilization and evolution in
bacteria.
Materials and methods
Genomic sequences and resources
Both completely and almost completely sequenced
bacterial genomes were downloaded from the ftp site
of National Center for Biotechnology Information
(NCBI) (ftp://ftp.ncbi.nlm.nih.gov/genomes/). A total
of 5207 genomes were retrieved (as of January 2014).
Information about environmental and other factors
(such as habitat, oxygen requirement, optimal
growth temperature and Gram staining) associated
with these organisms was acquired from either
NCBI or Genomes OnLine Database (GOLD) (Reddy
et al., 2015).
Identification of Se utilization traits in different
organisms
To analyze the occurrence of different Se traits, we
used Escherichia coli SelD, SelA and SelB sequences
as queries to search for components of the Sec trait
(Zhang et al., 2006), SelD and YbbB for the SeU trait
(Wolfe et al., 2004; Zhang et al., 2006), and SelD,
YqeB and YqeC for the Se-cofactor trait (Zhang et al.,
2008). TBLASTN (Altschul et al., 1990) was initially
used to identify genes encoding homologs with a
cutoff E-value of 0.01 and the alignment coverage of
at least 30%. Orthologous proteins were defined
using the conserved domain database (Pfam or CDD)
(Bateman et al., 2002; Marchler-Bauer et al., 2015)
and bidirectional best hits (Wolf and Koonin, 2012).
If necessary, orthologs were also confirmed by
genomic location or phylogenetic analyses. The
distribution of SelD and the three known Se
utilization traits in different bacterial taxa was
presented by using the online Interactive Tree Of
Life (iTOL) tool (Letunic and Bork, 2007, 2011) based
on a recently developed phylogenetic tree of life
(Ciccarelli et al., 2006).
Multiple sequence alignment and phylogenetic analysis
Standard approaches were used to reconstruct
phylogenetic trees of each component of Se utilization traits. Multiple sequence alignments were
performed using CLUSTALW (Thompson et al.,
1994) with default parameters. Phylogenetic trees
of protein families were reconstructed by PHYLIP
programs (Felsenstein, 1989) using neighbor-joining
method, and were further evaluated by MrBayes
(Bayesian estimation of phylogeny) tool (Ronquist
and Huelsenbeck, 2003). The vector graphics editor
Inkscape software (version 0.91) (Minatani, 2015)
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was used to further refine the fonts and colors of the
phylogenetic trees.
Identification of selenoproteomes in all Sec-utilizing
organisms
We collected representative sequences for all bacterial selenoprotein families identified or predicted
before (Kryukov and Gladyshev, 2004; Zhang and
Gladyshev, 2005, 2008, 2010b; Zhang et al., 2005).
These sequences were then used to search against
the genomic sequences of all Sec-utilizing organisms
for selenoprotein homologs with TBLASTN (e-value
⩽ 0.01). The redundant selenoprotein sequences
were removed using a custom script program. The
presence of a putative Sec-encoding UGA codon
and a downstream SECIS element was also
analyzed using the bSECISearch tool (Zhang and
Gladyshev, 2005).
Metagenomic analysis of Se utilization
A total of 60 bacterial metagenomic data sets derived
from different ecological environments (marine,
freshwater, terrestrial and host-associated; 15 data
sets for each) were retrieved from the Joint Genome
Institute Metagenome/Integrated Microbial Genomes
(JGI-M/IMG) database (Markowitz et al., 2012).
To identify each Se utilization trait in different
environmental samples, we used a strategy that was
similar to what we had done for sequenced organisms (see above). In each sample, if the occurrence of
genes involved in the same Se utilization trait was
different, the smallest number was used to represent
the frequency of the corresponding Se trait. The
recombinase A (recA) gene, which is a highly
conserved housekeeping gene and has been widely
used in many comparative metagenomic studies
(Handelsman, 2004; Wu et al., 2011), was used for
normalization of the occurrence of each gene in
samples.
Results
Generation of the largest map of Se utilization in
bacteria
Very recently, we have analyzed the distribution of
key components involved in each Se utilization
machinery, including Sec (SelD/SelA/SelB), SeU
(SelD/YbbB) and Se-cofactor (SelD/YqeB/YqeC)
traits, in more than 2000 bacterial genomes (Lin
et al., 2015). In this study, we extended such analysis
to more than 5200 bacterial genomes, which has
generated the largest and the most precise Se
utilization map in bacteria thus far. Figure 1 represents the distribution patterns of SelD and three
Se utilization traits in different bacterial phyla or
clades (details of the occurrence of SelD and other
key components involved in each Se utilization trait
are shown in Supplementary Table S1).
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It has been proposed that SelD protein was
required for all Se utilization traits, which defined
the overall Se utilization. In this study, except for
phyla containing one or two sequenced genomes
(such as Dictyoglomi, Elusimicrobia and Lentisphaerae), SelD was detected in almost all bacterial
lineages with the exception of a small number of
phyla or clades such as Chlamydiae, Mollicutes,
Deinococcus-Thermus and Thermotogae. This
implies that Se utilization is an ancient trait that
once was common to almost all species in bacteria.
However, only 1754 organisms (33.7% of all
sequenced bacteria) were detected to contain SelD,
suggesting that most species have lost the ability to
use this micronutrient over the long process of
evolution. This is consistent with previous observations (Zhang et al., 2006; Lin et al., 2015).
Among all sequenced bacterial genomes, we
identified 1121 Sec-utilizing (21.5%), 980 SeUutilizing (18.8%) and 312 Se-cofactor-utilizing
(6.0%) organisms. SelB has been suggested to be
the signature of the Sec and YbbB of the SeU traits
(Romero et al., 2005). Our data showed a perfect
consistency between these two genes and the
occurrence of Sec or SeU traits. Because of the
common component SelD, significant overlaps could
be observed between different Se utilization traits
(Figure 2). For example, 96 organisms were found to
possess all known Se utilization traits. Hypergeometric test suggested a significant relationship
between any two of the three Se utilization traits
(P-value o0.05). All these results were consistent
with our previous assumption that the presence of
one Se trait may be beneficial to acquisition of
others, probably partially due to the presence of SelD
(Zhang et al., 2006; Lin et al., 2015).
Some organisms were found to have highly similar
homologs of certain proteins (for example, SelA,
YbbB, YqeB or YqeC), but lack a complete Se
utilization
trait
(details
are
shown
in
Supplementary Table S1). Considering that the
majority of them have not been fully sequenced or
assembled, the possibility that other known genes
were not sequenced could not be neglected. It is also
possible that some of these homologs are involved in
Se-independent processes in certain organisms. The
hypothesis that SelA may have additional function
has been proposed before (Zhang et al., 2006).
Very recently, it was suggested that an alternative
enzymatic function might be hidden in the SelA
protein in Helicobacter pylori that does not use
Se (Cravedi et al., 2015). In addition, only the
co-occurrence of YqeB and YqeC proteins could be
used as a signature for the Se-cofactor utilization trait,
implying that each of them may participate in some
processes that are unrelated to Se (Zhang et al., 2008).
Identification of organisms containing orphan SelD
Previously, we have noticed the presence of very few
organisms that had orphan SelD (containing SelD but
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Figure 1 Distribution of SelD and known Se utilization traits in bacteria. The phylogenetic tree is simplified to only show major bacterial
taxa and branches. The four tracks (circles) around the tree (from inside to outside) represent the distribution patterns of SelD, Sec trait,
SeU trait and Se-cofactor trait, respectively. The length of the colored section of each column represents the percentage of organisms that
have either SelD or the corresponding Se trait among all sequenced organisms in this phylum: purple, SelD; red, Sec trait; green, SeU trait;
pink, Se-cofactor trait; grey, the rest of sequenced organisms. pro. stands for proteobacteria.
Figure 2 Venn diagram representation of the distribution of Se
utilization traits in bacteria. The number of organisms containing
the corresponding Se traits is indicated. The number of organisms
that contain orphan SelD is highlighted in red.
lacking any other known components of Se utilization traits and selenoprotein genes) (Lin et al., 2015).
With the largest number of sequenced bacterial
genomes retrieved in this study, we identified at
least 26 orphan SelD-containing organisms (Table 1).
These organisms belong to several evolutionarily
distant bacterial phyla or subdivisions, such as
Actinopolyspora iraqiensis IQ-H1 (Actinobacteria),
Brevundimonas sp. BAL3 and Methylobacterium
nodulans (Alphaproteobacteria), Xenorhabdus bovienii
and X. nematophila (Gammaproteobacteria/Enterobacteriales) and Prochlorococcus sp. W7 ~ W9
(Cyanobacteria). It is most likely that a novel and
unknown SelD-based Se utilization trait is present in
at least some of these organisms. On the other hand,
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Table 1 The list of organisms that contain orphan SelD
Phyla
Organism
Actinobacteria
Actinobacterium SCGC AAA015-D07
Actinomadura atramentaria DSM 43919
Actinopolyspora iraqiensis IQ-H1
Amycolatopsis vancoresmycina DSM 44592
Saccharomonospora halophila 8
Saccharomonospora saliphila YIM 90502
Streptomyces ghanaensis ATCC 14672
Bacteroidetes bacterium SCGC AAA027-G08
Prochlorococcus sp. W7
Prochlorococcus sp. W8
Prochlorococcus sp. W9
SAR406 cluster bacterium JGI 0000059-D20
SAR406 cluster bacterium JGI 0000059-E23
SAR406 cluster bacterium SCGC AAA003-E22
candidate division EM 19 bacterium SCGC AAA471-D06
candidate division KSB1 bacterium SCGC AAA252-G07
candidate division KSB1 bacterium SCGC AAA252-N05
Brevundimonas sp. BAL3
Methylobacterium nodulans ORS 2060
SAR324 cluster bacterium SCGC AAA001-C10
SAR324 cluster bacterium SCGC AAA240-J09
Xenorhabdus bovienii SS-2004
Xenorhabdus nematophila ATCC 19061
Gallaecimonas xiamenensis 3-C-1
Pseudomonas tolaasii 6264
Haemophilus aegyptius ATCC 11116
Bacteroidetes
Cyanobacteria
Others
Proteobacteria/alpha/others
Proteobacteria/delta
Proteobacteria/gamma/Enterobacteriales
Proteobacteria/gamma/Others
Proteobacteria/gamma/Pseudomonadales
Proteobacteria/gamma/Pasteurellales
the possibility that some of these selD genes are
either pseudogenes or involved in non-Se-related
processes could not be fully excluded.
To investigate possible genes that might be
involved in this unknown SelD-related trait, we
examined 10 genes upstream and downstream of
selD in these organisms. Three genes were chosen as
SelD-related candidates due to their genomic locations close to selD in at least three organisms
belonging to different phyla, including genes encoding an isochorismatase-like protein (pfam00857),
ABC transporter-related ATPase (pfam00005) and a
cysteine desulfurase-like (pfam00266) protein
(Supplementary Figure S1). Specific functions of
these proteins are not clear yet. Future experiments
are needed to verify the expression of orphan selD
genes in the corresponding organisms and to study
the relationship between these proteins and SelD or
Se metabolism.
Identification of new horizontal gene transfer events for
all Se utilization traits
Previous comparative studies of Se utilization have
reported several horizontal gene transfer (HGT)
events for both Sec and SeU traits, such as HGTs of
the entire Sec utilization pathway observed between
Treponema denticola (Spirochaetes) and Photobacterium profundum (Gammaproteobacteria), as well
as between Pseudomonadale species (Gammaproteobacteria) and Betaproteobacteria (Romero et al.,
2005; Zhang et al., 2006). In this study, we
reconstructed the phylogenetic trees for the key
components of each known Se trait based on
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thousands of bacterial species. In spite that most
branches were consistent with the evolutionary
relationship among bacterial species, we identified
new HGT events for each Se utilization trait,
including the Se-cofactor trait that had not been
reported so far.
Table 2 shows the HGT events that may have
happened very recently, which could be supported
by the widespread occurrence of the Se trait in many
species of the donor-containing phyla and the
absence of the same trait in all or almost all
sister species of the recipient organism. For
example, among all sequenced Marinobacter species
(Gammaproteobacteria/Alteromonadales), M. daepoensis
DSM 16072 was the only organism that contains
the Sec trait, which might have acquired
from Pseudomonas species (Gammaproteobacteria/
Pseudomonadales). Moreover, in the majority of both
donor and recipient organisms, the key genes
(including selD) involved in the corresponding Se
utilization traits as well as the only selenoprotein
gene (formate dehydrogenase alpha subunit) in those
Sec-utilizing organisms are either very close or even
organized in single operons.
Here, we identified HGT events for the Se-cofactor
trait in nature even though it was very rare. The
phylogenetic tree topologies of both YqeB and
YqeC revealed that the Fusobacterium sp. 11_3_2
(Fusobacteria) sequences were within the Firmicutes/
Clostridia node, and not within the Fusobacteria node
as expected by vertical descent (Figure 3a). Similar
HGT was also observed for Treponema phagedenis
F0421 (Spirochaetes), which also acquired the Secofactor trait from Firmicutes/Clostridia (Figure 3b).
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Table 2 New HGT events for each Se utilization trait
Se trait
From (donor)
Sec
Microlunatus phosphovorus NM-1
(Actinobacteria/Propionibacterineae)
Nakamurella multipartita DSM 44233
(Actinobacteria/Frankineae)
Pseudomonas stutzeri A1501
(Gammaproteobacteria/Pseudomonadales)
Aeromonas hydrophila pc104A
(Gammaproteobacteria/Aeromonadales)
SeU
Bacillales species (Firmicutes/Bacillales)
Se-cofactor Clostridium species (Firmicutes/Clostridia)
Clostridium species (Firmicutes/Clostridia)
To (recipient)
Occurrence of
other Se trait
in recipient
Arthrobacter crystallopoietes BAB-32 (Actinobacteria/
Micrococcineae)
Arthrobacter sp. 162MFSha1.1 (Actinobacteria/Micrococcineae)
—
Marinobacter daepoensis DSM 16072 (Gammaproteobacteria/
Alteromonadales)
Psychromonas sp. CNPT3 (Gammaproteobacteria/
Alteromonadales)
Lacticigenium naphtae DSM 19658 (Firmicutes/Lactobacillales)
Fusobacterium sp. 11_3_2 (Fusobacteria)
Treponema phagedenis F0421 (Spirochaetes)
SeU
—
SeU
—
—
—
Abbreviation: HGT, horizontal gene transfer.
Thus, HGT events appeared to have contributed to
the evolution of all Se utilization traits. However, so
far we could not identify one HGT event for the cotransfer of more than one Se utilization traits.
One previous hypothesis was that in the presence
of SelD that has been used by one Se trait,
acquisition of an additional Se trait via HGT might
be easier during evolution (Zhang et al., 2006). Here,
more HGT events were observed in organisms that
lack any other Se utilization traits (Table 2). This
implies that the pre-existing selD gene is not a strong
factor that could influence the occurrence of HGT,
although less Se-related genes are needed to be
laterally transferred from other organisms. However,
considering that the HGT events observed for all Se
utilization traits are very rare, the advantage of an
already existing selD gene for acquiring other Se
traits by HGT is not clear yet.
Identification of selenoproteomes in all sequenced Secutilizing organisms
As mentioned above, Sec is the major biological form
of Se and is incorporated into selenoproteins to
participate in a variety of redox and metabolic
processes. To better understand the utilization and
function of Se, it would be important to characterize
the selenoproteomes in different organisms. Here, we
have collected all known and previously predicted
prokaryotic selenoprotein families reported to date
(B90 families or subfamilies) and used representative sequences for each of them to search for Seccontaining homologs in all Sec-utilizing organisms
(see Materials and methods). The number of examined organisms was eight times more than previous
studies (Zhang and Gladyshev, 2010b).
The occurrence and size of selenoproteomes in
different bacterial phyla or clades are shown in
Figure 4 (details are shown in Supplementary
Table S2). Two bacterial phyla, Deltaproteobacteria
and Firmicutes/Clostridia, have been previously
considered as selenoprotein-rich phyla in which
the
majority
of
sequenced
species
were
selenoprotein-rich organisms (that is, containing at
least six selenoprotein genes as defined before)
(Zhang et al., 2006; Zhang and Gladyshev, 2009,
2010b). On the basis of current results, much more
selenoprotein-rich organisms could be identified in
these two phyla, including several new organisms
that have more than 30 selenoprotein genes
(Supplementary Table S2). To date, the largest
selenoproteome in sequenced bacteria has been
found in Syntrophobacter fumaroxidans (Deltaproteobacteria), which contains at least 39 selenoprotein
genes (Zhang and Gladyshev, 2010b). Interestingly,
we identified a new selenoprotein-rich phylum:
Synergistetes. In this phylum, 13 out of 14 sequenced
organisms (92.9%) could use Sec and at least 10
organisms (71.4%) were selenoprotein-rich organisms (Figure 4). It should be noted that a small
number of Sec-utilizing organisms appeared to lack
known selenoprotein genes, most likely due to their
incomplete genomes. In addition, the possibility that
new selenoprotein genes are present in some of these
organisms could not be excluded. Further investigation of all known selenoprotein families revealed
that the majority of them could be identified in
currently sequenced bacteria. Distribution of
sequences and organisms for the top 20 selenoprotein families is shown in Table 3 (more details are
shown in Supplementary Table S3). Formate dehydrogenase alpha subunit and SelD were the most
widespread selenoproteins in bacteria, which is
consistent with previous observation (Zhang et al.,
2006). We also measured the fraction of selenoprotein genes in all completely sequenced Sec-utilizing
organisms. No significant correlation could be
observed between the total number of selenoprotein
genes and that of all genes annotated in the
corresponding genomes (Supplementary Figure S2).
Thus, our data greatly extended previous analysis by
examining additional sequenced genomes and provided new information on the complex evolutionary
process of selenoproteins in bacteria.
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Figure 3 New HGT events revealed by phylogenetic analysis of YqeB and YqeC sequences. Organisms from different phyla or clades are
shown in different colors. (a) HGT between Firmicutes/Clostridia and Fusobacterium sp. 11_3_2 and (b) HGT between Firmicutes/
Clostridia and T. phagedenis F0421. Red: Firmicutes/Clostridia, blue: Firmicutes/Lactobacillales, purple: Gammaproteobacteria/
Enterobacteriales, green: Fusobacteria, pink: Spirochaetes. The branch lengths and bootstrap values are also shown.
Re-investigation of the relationship between Se
utilization and environmental factors
Previously, by using a limited number of organisms,
we tried to analyze the relationship between the Sec
and/or the SeU traits and different factors (for
example, habitat, oxygen requirement, optimal temperature, geographical location, Gram strain and GC
content). It was suggested that the Sec utilization
trait might favor anaerobic and/or hyperthermic
conditions, whereas the SeU trait might favor aerobic
and mesophilic conditions (Zhang et al., 2006;
Zhang and Gladyshev, 2010b). Considering the
number and distribution bias of sequenced bacterial
genomes at that time, it is necessary to recheck such
relationship using much more sequenced organisms
available now.
In this study, we collected the information about
ecological environments and some other factors for
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all organisms, and analyzed the contribution of each
of these factors for organisms containing Sec, SeU
and/or Se-cofactor traits. First, we found that both
Sec and Se-cofactor traits favored a host-associated
condition, whereas organisms possessing the SeU
trait were aquatic species that were mostly isolated
from sea or freshwater (Figure 5). Second, in
contrast with the previous observation (Zhang
et al., 2006), oxygen requirement analysis revealed
that Sec-utilizing organisms appeared to favor both
aerobic and anaerobic conditions (40.4% and 35.8%,
respectively; Figure 6). Thus, our previous hypothesis that anaerobic condition was a factor promoting
the use of Sec might be biased, most likely due to the
insufficient number of genomes of aerobic organisms
at that time. With a significant increase in the
number of sequenced aerobic species in the recent
decade, our results should be more reasonable to
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Figure 4 Occurrence and composition of selenoproteomes in bacteria. The majority of selenoprotein-rich organisms (containing at least
six selenoprotein genes) belong to three phyla, which are highlighted in red: Deltaproteobacteria, Firmicutes/Clostridia and Synergistetes.
Table 3 Distribution of the top 20 selenoprotein families in bacteria
Selenoprotein family
Formate dehydrogenase alpha subunit
Selenophosphate synthetase
Glycine reductase complex selenoprotein B
Glycine reductase complex selenoprotein A
Proline reductase
HesB like
Peroxiredoxin (Prx)
Coenzyme F420-reducing hydrogenase delta subunit
Heterodisulfide reductase subunit A
DsbA-like protein
Arsenite S-adenosylmethyltransferase
Selenoprotein W like
Fe-S oxidoreductase
Prx-like thiol:disulfide oxidoreductase
Coenzyme F420-reducing hydrogenase, alpha subunit
UGSC-containing protein
ULPU-containing selenoprotein
Cation-transporting ATPase, E1-E2 family
Predicted NADH:ubiquinone oxidoreductase, subunit RnfC
Sulfurtransferase homologous to a rhodanese-like protein
show a general trend of Sec utilization in bacteria.
However, the fact that more than three-fourths of
selenoprotein-rich organisms (78.3%) were anaerobic species suggested a somewhat more complex
relationship between selenoproteins and low oxygen
level. The majority of SeU-utilizing organisms
(53.4%) favored aerobic environments (Figure 6),
which is consistent with our previous observation.
On the other hand, almost four fifths (79.8%) of the
Se-cofactor-utilizing organisms lived in anaerobic
environment (Figure 6). Thus, it appears that
anaerobic conditions could significantly promote
the use of the Se-cofactor trait, which has never
been reported before. A more detailed analysis of the
correlation between oxygen requirement and different Se traits is shown in Supplementary Figure S3A.
Number of organisms
Number of selenoproteins
822
383
146
142
110
86
78
53
42
41
39
35
31
28
26
26
26
25
25
24
1276
394
261
165
131
90
91
131
103
45
41
35
47
32
31
33
28
32
28
25
Finally, temperature seemed to be an important
factor that could affect different Se utilization traits.
The Sec trait favored thermophilic conditions,
whereas psychrophilic bacteria appeared to prefer
the SeU trait (Supplementary Figure S3B), which
basically agreed with previous results (Zhang et al.,
2006). No significant correlation could be observed
between temperature and the Se-cofactor trait. Other
factors, such as geographical location, Gram strain
and GC content, had no significant effect on the
evolution of Se utilization.
Metagenomic analysis of Se utilization traits
To further verify the relationship between Se
utilization patterns and the habitat/environmental
The ISME Journal
Comparative genomics of selenium utilization
T Peng et al
2056
Figure 5 Relationship between Se utilization traits and habitat. Five types of habitat were analyzed, including host-associated, aquatic,
terrestrial, multiple and specialized.
organisms in almost all environmental samples
examined here, which is generally consistent with
the above observation (Supplementary Figure S4).
Based on identification of the occurrence of genes for
each Se utilization trait in each environmental
sample data set, we found that the Sec utilization
trait was more frequently present in host-associated
and aquatic (especially marine) habitats, whereas the
SeU and Se-cofactor traits were significantly
enriched in aquatic (either marine or freshwater)
and host-associated bacterial communities, respectively (Figure 7). This is also consistent with what we
have observed in sequenced genomes. Therefore, our
results provide new insights into the global trend in
microbial Se utilization in different ecological
environments.
Figure 6 Relationship between Se utilization traits and oxygen
requirement. Four types of oxygen requirement were analyzed,
including anaerobic, facultative, microaerophilic and aerobic.
preferences of bacteria, we performed a comparative
metagenomic analysis of Se utilization in 60 bacterial communities from different habitats or environments: aquatic (marine and freshwater), terrestrial
and host-associated. Sample details are shown in
Supplementary Table S4.
Analysis of the occurrence of selD gene revealed
that it might not be essential for the majority of
The ISME Journal
Discussion
The importance of Se in the physiology of both
prokaryotes and eukaryotes has been well established (Rayman, 2000; Hatfield et al., 2006).
Although much effort has been placed on characterizing pathways that Se is involved in and the
function of selenoproteins (Forchhammer and
Böck, 1991; Kryukov et al., 2003; Zhang and
Gladyshev, 2008, 2009; Zhang et al., 2008), the
evolution and ecology of Se utilization traits remains
largely unknown. Some considerations about the
evolutionary patterns of different Se traits and
Comparative genomics of selenium utilization
T Peng et al
2057
Figure 7 Metagenomic analysis of the relationship between Se
utilization and different environments. Samples were collected
from four types of habitat: host-associated, marine (aquatic),
freshwater (aquatic) and terrestrial.
selenoprotein families have previously been proposed (Zhang et al., 2006; Castellano, 2009; Zhang
and Gladyshev, 2009, 2010b). However, due to much
less genomic resources available at that time, it is
unclear whether these observations or hypotheses
could correctly reflect the general distribution and
evolutionary dynamics of Se utilization over the
whole bacterial domain. In the present study, we
greatly extended such analysis for all known Se
utilization traits to more than 5200 bacterial species,
which was eight times larger than previous analysis
(Zhang and Gladyshev, 2010b). Our data provide the
largest and most comprehensive view of Se utilization in bacteria. Such strategy and resources
collected here can be easily extended to analyze
the utilization and evolution of many other micronutrients such as metals.
Comparative genomics analysis of known Se
utilization traits revealed that Se is an important
element for a variety of organisms in almost all
bacterial phyla. However, among all sequenced
bacterial organisms, only one-third of organisms
had at least one Se trait, implying that most bacterial
lineages lost the ability to use this element. Previous
hypothesis that different Se traits could affect
evolution of each other (Zhang et al., 2006; Lin
et al., 2015) has been further confirmed by analyzing
more bacterial genomes.
As SelD is a common feature for all Se utilization
pathways, identification of several species that
possess orphan selD gene raised the possibility
of a new SelD-related Se utilization trait in these
organisms. By examining several genes upstream
and downstream of selD gene in each of those
genomes, three of them were considered as candidates that might be functionally related to SelD,
including isochorismatase-like, ABC transporterrelated ATPase and cysteine desulfurase-like proteins. Future challenges would be to experimentally
verify the new SelD-dependent Se form and identify
the function of these genes.
It is known that HGT events can contribute to the
evolution of a variety of biological processes,
including Se utilization (Romero et al., 2005;
Zhang et al., 2006). Although HGT of the entire Se
utilization trait is difficult, we observed additional
cases besides the HGT events previously reported for
the Sec and SeU traits. For the first time, we
identified HGT for the Se-cofactor trait, which has
never been reported before. Moreover, identification
of more HGT events in the organisms that lack any
other Se traits suggests that an already existing selD
gene used by one Se trait might not be strongly
needed by HGT of additional Se traits in certain
organisms.
There is no doubt that, in this study, identification
of selenoproteomes in all Sec-utilizing organisms has
generated the largest data set of selenoproteins in
this domain of life. These data revealed new
organisms that have a large number of selenoproteins
and a new selenoprotein-rich phylum: Synergistetes,
which provide novel insights into the evolution of
selenoproteins in bacteria. In addition, newly identified selenoprotein-rich organisms may have farreaching implications for their potential use for
the development of Se-enriched bio-products.
For example, several Se-enriched algae (such as
Palmaria Palmata) have been regarded as the best
alternatives to cereals in food and feed (Maehre et al,
2014). In the future, it would be valuable to
investigate the evolutionary dynamics of different
selenoprotein families and the complex crosstalk
among them.
An additional significant contribution of this work
is that re-investigation of the relationship between
environmental factors and Se utilization suggests
new ecological features of each Se utilization trait.
The Sec and Se-cofactor traits favor a host-associated
condition, whereas the SeU trait favors aquatic
environments. These Se utilization patterns have
been verified by comparative metagenomic analysis
of distinct bacterial communities from different
habitats/environments around the world. The reason
for such a trend is not clear yet. One possibility
might be that host-associated (such as bacterial
parasites in animals) and aquatic (especially marine)
environments could provide sufficient supply of Se.
However, Se utilization in terrestrial bacteria may be
heavily dependent on diverse Se levels in different
areas. In addition, the substantial microbial taxonomic diversity in different ecological environments
might also explain differences in Se utilization.
Surprisingly, we could not identify the strong
relationship previously reported between oxygen
level and Sec utilization (Zhang et al., 2006; Zhang
and Gladyshev, 2010b), as a significant number of
both aerobic and anaerobic organisms have this
trait. However, considering that the majority of
selenoprotein-rich organisms are anaerobic organisms, low oxygen level might, at least in part,
contribute to the evolution of new selenoprotein
genes. On the other hand, a significant correlation
The ISME Journal
Comparative genomics of selenium utilization
T Peng et al
2058
between the Se-cofactor trait and anaerobic conditions demonstrated a strong macroevolutionary trend
of this trait for the first time. In the future, it would
be important to identify additional ecological factors
that influence Se utilization in nature.
In conclusion, we have performed a comprehensive comparative analysis of Se utilization in
bacteria, which generated the largest map of Se
utilization in this domain. Our data revealed a
complex and dynamic evolutionary history for each
Se utilization trait. Identification of organisms that
only have SelD implies the presence of a new SelDdependent Se utilization pathway in bacteria.
Phylogenetic analyses of key components of Se
utilization traits suggested that additional HGT
events could be detected for each of them; however,
co-transfer of two or more Se traits might be
extremely difficult and has not been observed yet.
Identification of new selenoprotein-rich phylum and
selenoprotein-rich organisms as well as their selenoproteomes may provide important perspectives on
the diversity and evolution of different selenoprotein
families. Finally, comparative analyses of environmental conditions for all sequenced bacteria and
metagenomic samples revealed new and important
relationships between ecological environments and
Se utilization.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China under No. 31171233, and a grant from
Chinese Academy of Sciences (CAS) (2012OHTP10).
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